A gram-positive coccus, Staphylococcus aureus is one of the most important human pathogens. S. aureus causes a variety of infections with high morbidity and mortality including osteomyelitis, soft tissue infections, pneumonia, endocarditis, and brain abscesses. In the United States, there are approximately 292,000 hospital admissions of S. aureus infections annually. A survey showed that 19,000 people died in 2006 from S. aureus infections. To further intensify the scope of the problem, the rate of methicillin-resistant S. aureus isolates found both in the community and in hospitals is continuing to increase.
S. aureus is also a major cause of ocular infections, including blepharitis, conjunctivitis, keratitis, and endophthalmitis. Patients with these infections have intense pain, redness, and photophobia, and the infection can result in an ulcer with numerous infiltrating polymorphonuclear leukocytes. These infections can result in loss of visual acuity or blindness. Intensive antibiotic therapy can eradicate S. aureus from the infected site, but pathologic changes can continue due to action of toxic proteins that are secreted by S. aureus. The appearance of an infected eye may not be a good measure of the success of antibiotic treatment because the activity of the secreted toxins is not directly affected by the antibiotic-mediated killing of the bacteria.
A S. aureus protein that mediates ocular tissue damage is α-toxin. α-toxin has also been proven to have an important role in brain and respiratory infections caused by S. aureus and is being proposed as a vaccine target for humans. α-toxin is a 33 kDa protein that forms a ring of seven α-toxin molecules that penetrate the host cell membrane forming a pore, causing cell lysis. α-toxin has also been demonstrated to cause cellular changes in human cells, upregulate inflammatory cytokines, cause calcium disruptions reducing host defenses, and cause apoptosis. Furthermore, α-toxin has been shown to be an important virulence factor in both rabbit and murine models of keratitis. When the α-toxin gene was mutated, the pathology associated with the infection was significantly reduced compared to the parent strain. However, when the mutated α-toxin gene in the knockout strain was complemented, the pathology was restored.
To date, there are no known inhibitors of α-toxin available to treat S. aureus infection, including ocular infections. There have been efforts to develop inhibitors of this important virulence factor including the use of steroids or steroid-like molecules. It has been demonstrated that α-toxin binds to caveolin, a protein present in lipid rafts. α-toxin has a caveolin-1 binding motif that, when removed, results in a nonhemolytic form of α-toxin. Peptides that mimic the caveolin-binding domain of α-toxin have been shown to limit the action of α-toxin on cell membranes. It has been shown that methyl-β-cyclodextrin (CD) can bind weakly to the pore of α-toxin heptamer. On introducing amino acid changes in the α-toxin, Gu et al. were able to increase the binding of CD. Furthermore, Vijayvargia et al. observed that cholesterol depletion of cells arrested the action of α-toxin and that chelation of cholesterol, using CD, helped retard the pore formation by α-toxin. Karginov et al. have demonstrated that α-toxin action can be inhibited by positively charged side groups chemically attached to CD.
Embodiments of the present invention show that a complex of cholesterol and CD (CD-cholesterol) can inhibit the action of α-toxin. The results show that CD-cholesterol complex can inhibit α-toxin action in vitro and in vivo.